Ribonucleotide reductase is responsible for regulating a balanced and continued supply to deoxyribonucleotide precursors required for DNA replication. As an approach to understanding the structural and functional basis for regulation of ribonucleotide reduction we have isolated and characterized a membrane complex containing ribonucleotide reductase proteins B1, B2 and thioredoxin. Unlike the solubilized form of reductase which shows dilution inactivation, the specific activity in the membrane fraction is independent of protein concentration. The functional significance and the integrity of this membrane reductase complex is also suggested by the co-purification of several other enzymes involved in deoxynucleotide metabolism, including thymidylate synthetase and DNA polymerase I. Present studies will continue characterization of the various enzyme activities copurifying with the ribonucleotide reductase-membrane fraction together with studies on any functional relations that exist between these proteins. The enzymatic behavior of the membrane associated form of reductase will be carried out using the crude membrane fraction as well as the B1 protein purified as a lipoprotein complex on dATP-sepharose. Thioredoxin, the hydrogen donor implicated in ribonucleotide reduction, has been isolated as a phosphoprotein. Sequence analysis demonstrated that the phosphorylated amino acid is the same cysteine involved in the hydrogen donor reaction. Physiological studies will investigate the implied role of phosphotioredoxin in phosphate transfer reactions.